EXPERIMENTAL STUDY
Addition of spironolactone to angiotensin-converting enzyme inhibition in heart failure improves endothelial vasomotor dysfunction
Role of vascular superoxide anion formation and endothelial nitric oxide synthase expression
Johann Bauersachs, MD*,*,
Marina Heck ,
Daniela Fraccarollo, PhD*,
Steven K. Hildemann, MD ,
Georg Ertl, MD*,
Martin Wehling, MD and
Michael Christ, MD
* Medizinische Klinik der Julius-Maximilians-Universität Würzburg, Germany
Institut für Klinische Pharmakologie, Fakultät für Klinische Medizin Mannheim, Universität Heidelberg, Germany
Pharmacia GmbH, Erlangen, Germany
Klinik für Innere Medizin, Kardiologie, Philipps-Universität Marburg, Germany
Manuscript received April 13, 2001;
revised manuscript received September 10, 2001,
accepted October 18, 2001.
* Reprint requests and correspondence: Dr. Johann Bauersachs, Medizinische Universitätsklinik, Josef-Schneider-Str. 2, D-97080 Würzburg, Germany.
j.bauersachs{at}medizin.uni-wuerzburg.de
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Abstract
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OBJECTIVES: We sought to investigate the effects of adding spironolactone (SP) to angiotensin-converting enzyme (ACE) inhibition on endothelium-dependent vasodilation in rats with chronic heart failure (CHF).
BACKGROUND: Adding SP to ACE inhibitors reduces mortality and morbidity in CHF. Endothelial vasomotor dysfunction contributes to increased peripheral vascular resistance and reduced myocardial perfusion in CHF.
METHODS: Seven days after extensive myocardial infarction (CHF) or sham operation, Wistar rats were treated with placebo, the ACE inhibitor trandolapril (TR, 0.3 mg/kg body weight per day), SP (10 mg/kg per day) or a combination of both for 11 weeks.
RESULTS: Maximal acetylcholine-induced, nitric oxide (NO)-dependent relaxation was significantly attenuated in aortic rings from rats with CHF as compared with sham-operated animals (Rmax 44 ± 3% vs. 63 ± 3%). Spironolactone alone had no influence (46 ± 5%) and TR improved NO-mediated relaxation (55 ± 4%), whereas treatment with both completely restored endothelium-dependent vasorelaxation (64 ± 4%). Aortic superoxide formation was significantly increased in rats with CHF as compared with sham-operated animals, but was normalized by treatment with SP or SP plus TR. In addition, aortic messenger ribonucleic acid expression of the oxidase subunit p22phox in rats with CHF was significantly reduced by SP or TR plus SP. Endothelial NO synthase expression was increased in TR-treated animals. Incubation of isolated porcine coronary arteries with SP dose-dependently attenuated superoxide formation.
CONCLUSIONS: Spironolactone added to an ACE inhibitor normalizes NO-mediated relaxation in experimental CHF by beneficially modulating the balance of NO and superoxide anion formation.
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Abbreviations and Acronyms
| | ACE | | angiotensin-converting enzyme | | CHF | | chronic heart failure | | eNOS | | endothelial nitric oxide synthase | | MI | | myocardial infarction | | NO | | nitric oxide | | O2– | | superoxide anion | | RT-PCR | | reverse transcription polymerase chain reaction | | SP | | spironolactone | | TR | | trandolapril |
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The endothelium plays a crucial role in the control of vascular tone by releasing endothelium-derived autacoids, the most important of which is nitric oxide (NO) (1). A reduced bioavailability of NO has been reported in cardiovascular diseases such as arterial hypertension, arteriosclerosis and diabetes (2), supporting the important role of NO in cardiovascular pathophysiology. In chronic heart failure (CHF), a reduced bioavailability of NO in the peripheral and coronary arteries increases peripheral vascular resistance and reduces myocardial perfusion, contributing to reduced myocardial function (3–7). As oxidative stress is increased in patients with CHF (8,9) and superoxide anion (O2–) rapidly scavenges NO in the vascular wall, a reduction of bioactive NO may occur, despite normal or even increased NO generation (10). Indeed, endothelial dysfunction coincided with an increase in vascular endothelial NO synthase (eNOS) expression and a concomitant increase in O2– generation, leading to a reduced NO bioavailability in experimental CHF (11).
Mortality of patients with CHF has been reduced by treatment with angiotensin-converting enzyme (ACE) inhibitors, which may be explained, at least in part, by an improvement of endothelial function and subsequent improvement of perfusion and exercise capacity (12–14). However, morbidity and mortality of patients with CHF remain high. Increased plasma levels of aldosterone are considered to be an independent risk factor for a worse outcome in patients with CHF (15). Originally, it has been assumed that ACE inhibitors block angiotensin II–dependent adrenal aldosterone secretion. However, an "aldosterone escape" occurs (16). The clinical relevance of this observation has been supported by the convincing results of the recently published Randomized ALdactone Evaluation Study (RALES), in which the mineralocorticoid receptor antagonist spironolactone (SP) was added to ACE inhibition in patients with severe CHF, markedly reducing overall mortality by 30% (17) and improving endothelium-dependent NO formation (18). However, the mechanisms involved in the beneficial effects of SP on endothelium-dependent dilation remain unknown.
Therefore, we investigated the effect of long-term treatment with low-dose SP (19) and the ACE inhibitor trandolapril (TR), either alone or in combination, on endothelium-dependent and -independent relaxant responses in the aorta of rats with CHF after experimental myocardial infarction (MI). In addition, the formation of aortic O2– and the expression of p22phox, the critical subunit of NADPH oxidase, and eNOS were assessed.
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Methods
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Study protocol, MI and hemodynamic measurements.
Left coronary artery ligations were performed in adult male Wistar rats weighing 250 to 300 g, as previously described (20). Briefly, the thorax was opened under ether anesthesia; the heart was exteriorized; and a ligature was placed around the proximal left coronary artery. On the seventh postoperative day, surviving rats were randomly allocated to placebo, the ACE inhibitor TR (0.3 mg/kg body weight per day), SP (10 mg/kg per day) or a combination of TR and SP. Hemodynamic studies were performed 12 weeks after coronary artery ligation under barbiturate anesthesia and controlled respiration (20). Drug treatment was withheld for 24 h before hemodynamic studies to exclude acute drug effects.
Sample collection, determination of infarct size and ventricular remodeling.
After hemodynamic measurement, the heart was flushed in ice-cold Krebs-Ringer solution, and the left ventricle was the cut into three transverse sections: the apex, middle ring (3 mm) and base. From the middle ring, 5-µm sections were cut at 100-µm intervals and stained with picrosirius red. The boundary length of the infarct-related and non–infarct-related surfaces of the endocardium and epicardium were traced with a planimeter digital image analyzer, and infarct size (fraction of the infarct-related left ventricle) was expressed as a percentage of length. Only rats with extensive infarcts (>40%) were included in the study of vascular reactivity.
Vascular reactivity studies.
The descending thoracic aorta was dissected and cleaned of connective tissue. The upper section (10 mm) was immediately frozen for Western blot and polymerase chain reaction (PCR) analysis. The lower section (10 mm) was used for measurement of O2– production, whereas the remainder was cut into 3-mm rings, which were mounted in an organ bath (FMI, Seeheim, Germany) for isometric force measurements. The rings were equilibrated for 30 min under a rest tension of 2 g in oxygenated (95% oxygen and 5% carbon dioxide) Krebs-Henseleit solution (measurements in mmol/l: NaCl 118, KCl 4.7, MgSO4 1.2, CaCl2 1.6, K2HPO4 1.2, NaHCO3 25, glucose 12; pH 7.4; 37°C) containing diclofenac (1 µmol/l) (21). The rings were repeatedly contracted by KCl (100 mmol/l) until reproducible responses were obtained. Thereafter, the rings were preconstricted with phenylephrine (0.3 to 1.0 µmol/l) to comparable constriction levels, and the relaxant response to cumulative doses of acetylcholine and sodium nitroprusside was assessed (22).
Porcine coronary arteries.
Coronary arteries of porcine hearts (obtained from a local slaughterhouse) were cautiously dissected free of the adhering fat and connective tissue. Small segments ( 3 to 4 mm in length) were cultured in phenol red–free medium 199 (cc pro, Neustadt, Germany) containing polymyxin B (5 µg/ml) and penicillin/streptomycin (10 IU/ml and 10 µg/ml, respectively). The rings were incubated with SP, RU486, progesterone, cortisol, aldosterone or vehicle alone (control) up to 48 h.
Measurement of O2– formation.
Vascular O2– formation was measured using lucigenin-enhanced chemiluminescence (21). The light reaction between O2– and lucigenin (5 or 250 µmol/l [23]) was detected by a luminometer (Wallac, Freiburg, Germany) during incubation of the rings in a HEPES-modified Krebs buffer (pH 7.40). The specific signal was expressed as counts per minute per milligram of dry weight of tissue (cpm/mg).
Western blot analysis.
Rat aortae were homogenized, and crude protein extracts (20 µg) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Endothelial NOS protein was detected using a specific antibody (Transduction Laboratories, Lexington, Kentucky). Enhanced chemiluminescence (Pharmacia Biotech, Freiburg, Germany) was visualized, and the signals were densitometrically determined by the National Institutes of Health (NIH) Image software (version 1.62).
Quantification of messenger ribonucleic acid (mRNA) expression by competitive reverse transcription (RT)-PCR.
Vascular mRNA expression of eNOS, p22phox and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was determined by competitive PCR. Total RNA was isolated using Trizol reagent (Life Technologies, Eggenstein, Germany). After RT (Advantage RT-for-PCR Kit, Clontech Inc., Palo Alto, California), first-strand complementary deoxyribonucleic acid (cDNA) was used as a template for PCR. Nonhomologous internal standards with primer templates that are recognized by the gene-specific primers were constructed (PCR MIMIC Construction Kit, Clontech Inc.), and cDNA was incubated with decreasing amounts of known standards and amplified with gene-specific primers. Products of PCR amplification were separated on 1.5% (wt/vol) agarose gel preparations, and expression levels of ethidium-stained products of the templates and standards were densitometrically determined using the NIH Image software (version 1.61). Absolute expression levels were calculated by comparison with the signals of equimolar amounts of the standard.
Materials.
All biochemicals were obtained from Sigma (Deisenhofen, Germany). Spironolactone was provided by Pharmacia (Erlangen, Germany), and TR by Knoll AG (Ludwigshafen, Germany).
Statistics.
Relaxant responses were given as percent relaxation relative to the preconstriction level. Data are expressed as the mean value ± SEM of n experiments with segments from different arteries. Statistical analysis was performed by one-way analysis of variance, followed by the Tukey-Kramer multiple comparisons test or the two-tailed Student t test for unpaired data, as appropriate. A p value <0.05 was considered statistically significant.
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Results
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Global variables.
Global variables of rats with CHF and sham-operated animals are shown in Table 1. Infarct sizes were comparable among the different experimental groups. Left ventricular systolic pressure was significantly lower in all groups of rats with CHF, whereas left ventricular end-diastolic pressure was markedly elevated.
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Table 1 Variables in Rats With Chronic Heart Failure at 12 Weeks After Myocardial Infarction, as Compared With Sham-Operated Animals
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Vasodilator responses in aortic rings.
Acetylcholine induced a concentration-dependent relaxation of the aortic rings preconstricted with phenylephrine. Receptor-dependent relaxation was reduced in the aortae of rats with heart failure after extensive MI (Fig. 1A and Table 2). Long-term treatment with SP alone had no effect on acetylcholine-induced relaxation, whereas TR slightly improved endothelium-dependent relaxation (Fig. 1A and Table 2). Treatment of rats with a combination of SP and TR led to restoration of the acetylcholine-induced relaxation in the aortic rings of rats with CHF (Fig. 1A and Table 2). The concentration-response curve of the endothelium-independent vasodilator sodium nitroprusside was not different between sham-operated animals and rats with CHF (Fig. 1B and Table 2). The concentration-response curve to sodium nitroprusside in the aortae of rats treated with SP alone was shifted to the right, whereas endothelium-independent relaxation was similar in all other treatment groups (Fig. 1B and Table 2).
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Figure 1 Relaxations induced by acetylcholine (A) and sodium nitroprusside (B) in the aortic rings of rats with congestive heart failure (CHF) at 12 weeks after myocardial infarction (solid circles, diamonds, triangles and squares), as compared with sham-operated animals (open circles). The animals were treated with either placebo (Plac; solid and open circles), trandolapril (TR) (0.3 mg/kg body weight per day; solid diamonds), spironolactone (SP) (10 mg/kg per day; solid triangles) or a combination of TR and spironolactone (SP) (solid squares). Data are presented as the mean value ± SEM from 8 to 10 separate experiments. **p < 0.01 CHF/placebo group vs. sham/placebo group and CHF/TR-SP group.
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Table 2 Acetylcholine- and Sodium Nitroprusside-Induced Relaxations in the Aortic Rings of Rats With Chronic Heart Failure at 12 Weeks After Myocardial Infarction, as Compared With Sham-Operated Animals
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Superoxide anion formation and p22phox expression in the rat aorta.
Formation of O2– was significantly increased in the aortae of rats with CHF as compared with sham-operated rats. Formation of O2– was reduced in the aortae of TR-treated rats, whereas generation of O2– was completely normalized in all rats treated with SP (measurements in cpm/mg—sham/placebo: 651 ± 32; CHF/placebo: 1,066 ± 119; CHF/SP: 591 ± 104; CHF/TR: 831 ± 85; CHF/TR+SP: 581 ± 79) and lucigenin, 250 µmol/l. Similar results were obtained in a separate series of experiments using 5 µmol/l of lucigenin (Fig. 2).
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Figure 2 Production of superoxide anion (O2–) (lucigenin 5 µmol/l) in the aortic rings of rats with congestive heart failure (CHF) at 12 weeks after myocardial infarction, as compared with sham-operated animals (for experimental groups, see Fig. 1). Data are presented as the mean value ± SEM from 8 to 10 separate experiments. **p < 0.01 CHF/placebo group vs. sham/placebo group. p < 0.01 CHF/spironolactone (SP) group and CHF/trandolapril (TR)-SP group vs. CHF/placebo group.
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Vascular mRNA expression of p22phox was significantly reduced in rats with CHF treated with SP alone or in combination with TR (2.1 ± 0.2 and 1.9 ± 0.3 attomol/µg RNA), as compared with the other groups (sham/placebo: 2.9 ± 0.2; CHF/placebo: 3.7 ± 0.3; CHF/TR: 3.2 ± 0.5 attomol/µg RNA; p < 0.05), whereas mRNA expression of GAPDH was not changed (sham/placebo: 51 ± 9; CHF/placebo: 54 ± 10; CHF/TR: 50 ± 4; CHF/SP: 53 ± 13; CHF/TR+SP: 54 ± 7 nmol/µg RNA).
Expression of eNOS in the rat aorta.
There was a trend toward increased mRNA expression of vascular eNOS in rats with CHF treated with TR, either alone or in combination with SP (5.1 ± 1.0 and 5.4 ± 1.1 attomol/µg RNA), as compared with the other experimental groups (sham/placebo: 4.1 ± 0.2; CHF/placebo: 4.4 ± 0.1; CHF/SP: 4.2 ± 0.2 attomol/µg RNA). Endothelial NOS protein expression was elevated in rats with CHF as compared with sham-operated rats, as previously shown (11). Furthermore, eNOS protein expression was significantly higher in rats with CHF treated with TR, alone or in combination with SP, whereas eNOS expression tended to be reduced in rats with CHF treated with SP alone (Fig. 3).
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Figure 3 Aortic endothelial nitric oxide synthase (eNOS) expression in rats with congestive heart failure (CHF) at 12 weeks after myocardial infarction (relative to the signals obtained in the sham-operated rats), as compared with sham-operated animals (for experimental groups, see Fig. 1). Densitometric analysis (bottom; n = 5 to 6) and a representative blot (top). Data are presented as the mean value ± SEM. *p < 0.05 vs. sham/placebo, CHF/placebo and CHF/spironolactone (SP) groups. TR = trandolapril.
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Direct actions of SP and steroids on vascular radical generation.
In porcine coronary artery segments, basal superoxide anion generation was 862 ± 75 cpm/mg (n = 67) after culturing them for 48 h (lucigenin, 250 µmol/l). The addition of the flavoprotein inhibitor diphenyleneiodonium (10 µmol/l; 65 ± 62 cpm/mg; n = 7) significantly reduced any lucigenin-detectable chemiluminescence, whereas the addition of the radical scavenger tiron (10 mmol/l; n = 6) completely abrogated it (data not shown). Co-incubation of rings with 10 nmol/l of SP for 48 h significantly attenuated O2– generation as compared with respective control rings (427 ± 82 vs. 715 ± 87 cpm/mg; n = 7). Similar results were obtained using 5 µmol/l of lucigenin (Fig. 4). A reduction of vascular O2– generation by SP was dose-dependent (median effective concentration [EC50]: 1 nmol/l), reaching near maximal effects at 10 nmol/l. Acute addition of NADH (100 µmol/l) significantly increased O2– formation, as compared with basal conditions (lucigenin, 5 µmol/l) (Fig. 4). NADH-stimulated O2– formation was significantly attenuated in rings co-incubated with SP (Fig. 4). Spironolactone did not affect O2– generation after short-term (30 min) incubation of coronary segments. Furthermore, adding SP directly to the test tube did not affect the chemiluminescence signal, excluding any direct quenching effect of the drug.
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Figure 4 Generation of superoxide anion (O2–) in porcine coronary arteries cultured for 48 h in the absence (open bars) or presence of spironolactone (10 nmol/l; closed bars). Generation of O2– was determined at baseline and after addition of 100 µmol/l nicotinamide adenine dinucleotide (NADH). *p < 0.05.
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Culture of coronary rings in either aldosterone (10 nmol/l; 1,169 ± 133 cpm/mg) or hydrocortisone (10 nmol/l; 996 ± 149 cpm/mg) did not modulate vascular O2– formation, as compared with control conditions (1,146 ± 152 cpm/mg; n = 16 to 19), whereas incubation in progesterone (10 nmol/l; 541 ± 70 cpm/mg) and RU486 (10 µmol/l; 509 ± 60 cpm/mg) significantly reduced O2– formation, as compared with respective control conditions (818 ± 147 cpm/mg; n = 19). Attenuation of O2– generation by progesterone or SP was not further modulated by co-incubation with RU486 (progesterone: 571 ± 76 cpm/mg; SP: 448 ± 59 cpm/mg; n = 19).
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Discussion
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In the present study, adding SP to ACE inhibitor treatment completely restored the attenuated endothelium-dependent relaxation in rats with experimental CHF, whereas SP alone did not modulate it, and TR alone only slightly improved the endothelium-dependent vasodilator response. We observed an upregulation of eNOS expression and a modest reduction of vascular O2– formation by the ACE inhibitor TR , whereas SP significantly reduced vascular O2– formation and p22phox expression. Spironolactone-induced effects on vascular O2– generation appear to be mediated by direct actions on vascular radical generation, as demonstrated in separate experiments in cultured rings of porcine coronary arteries.
NO formation and eNOS expression in CHF..
Impairment of endothelium-dependent, NO-mediated vasorelaxation has been repeatedly reported in patients with CHF (4,24), probably contributing to elevated vascular resistance. Comparable results have been obtained in a rat model of experimental CHF, where attenuation of endothelium-dependent vasodilation has been reported in various vascular beds (5,11,25,26). The decreased release of NO may be explained by downregulation of eNOS, the key enzyme of endothelial, receptor-dependent NO liberation. Indeed, reduced vascular eNOS expression has been reported in pacing-induced CHF in dogs and in monocrotaline-induced pulmonary hypertension and CHF in rats (27,28). However, in rats with ischemic heart failure after extensive MI, NO-mediated relaxations are blunted, despite an increase in aortic eNOS expression, presumably due to an increased generation of superoxide radicals (11), leading to a reduction of NO bioavailability.
Superoxide formation and NO-mediated relaxation in CHF..
A NAD(P)H-dependent vascular oxidase in the aortic wall has been identified as a major source of increased vascular O2– generation in rats with chronic MI (11). In the present study, we extend these findings, demonstrating enhanced expression of p22phox, which is considered to be the critical subunit for NAD(P)H-dependent oxidase activity (29). Elevated levels of angiotensin II upregulate vascular subunit expression and increase oxidase activity (30). As plasma renin activity, as well as tissue ACE activity, is markedly elevated in rats with chronic MI (31), vascular O2– generation in rats with CHF may presumably be induced by increased angiotensin II formation. Conforming to this interpretation, TR may improve endothelial function in our rat model of CHF by a reduction of angiotensin II–dependent O2– generation. However, SP alone induced an even more pronounced attenuation of vascular O2– generation, as compared with TR, but had no effect on acetylcholine-induced relaxation. An improvement of endothelial function may be masked by reduced smooth muscle responsiveness, as suggested by the rightward shift of the concentration response curve to sodium nitroprusside. The marked reduction of O2– generation and p22phox expression by SP, as compared with the modest effects of TR on O2– formation, suggests that vascular oxidant stress in CHF is not solely dependent on angiotensin II–dependent mechanisms in rats after extensive MI. Indeed, enhanced vascular O2– generation has also been observed in low-renin hypertension (32), suggesting that increased vascular O2– generation may be partially independent of angiotensin II.
Our data imply that ACE inhibition may improve endothelial vasomotor dysfunction by an increase in endothelial-dependent NO release, in addition to a reduction of O2– generation. Indeed, ramipril, at dosages that are not acting as antihypertensive, increases eNOS expression and NO synthesis in the carotid arteries and thoracic aortae of spontaneously hypertensive rats (33). Because only the combination of SP and TR was able to completely restore NO-mediated relaxation in rats with CHF, we suggest that both the marked increase in eNOS expression and activity and the normalization of O2– formation by SP may be crucial for the beneficial effect on NO/O2– balance.
Attenuation of vascular superoxide formation by SP.
One may speculate that increased plasma aldosterone levels, even during ACE inhibition, induce endothelial dysfunction by an increased formation of superoxide anions. A low-potassium diet markedly increases aortic superoxide formation in rats; thus, the potassium-sparing effect of SP may contribute to the reduction of O2– generation in rats with CHF (34). However, our rats were fed with standard chow, and plasma K+ was not elevated by SP in a comparable study setting (19). Furthermore, aldosterone may directly act on the vasculature and increase vascular radical generation. However, neither aldosterone nor cortisol increased O2– formation in isolated porcine coronary arteries, whereas SP significantly attenuated radical generation. Thus, an action of SP independent of mineralocorticoid receptor antagonism may mediate a reduction of aortic O2– formation in rats with CHF and in isolated porcine coronary arteries. This interpretation is supported by reduced O2– formation in rings co-incubated with progesterone. Those findings may point to involvement of the progestagenic actions of SP (35). However, we were not able to prove this hypothesis, as the progesterone receptor antagonist RU486 displays comparable inhibitory effects on radical generation.
Clinical implications.
The beneficial modulation of NO/O2– balance observed in our study provides an explanation for the improvement of endothelial function in patients with CHF treated with SP in addition to ACE inhibition (18). Thus, we speculate that SP may enhance tissue perfusion and exercise capacity in patients with CHF by a reduction of vascular O2– generation, in addition to the beneficial effects of ACE inhibition. A reduction of coronary O2– generation and subsequent improvement of endothelial function may beneficially modulate left ventricular remodeling, probably leading to a better quality of life and prognosis (7,36).
Conclusions.
In experimental CHF, combination therapy with SP and the ACE inhibitor TR restored endothelial function more efficiently than the ACE inhibitor alone. A better NO/O2– balance is probably achieved by a marked upregulation of eNOS expression by the ACE inhibitor TR and a significant reduction of O2– formation by SP. The beneficial actions of SP in CHF (as demonstrated in RALES) may be explained, at least in part, by the beneficial effects on vascular radical generation.
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Acknowledgments
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The authors thank Elke Kirsch, Claudia Liebetrau and Anna Dembny for their expert technical assistance, and Prof. H. Basler and Prof. H. Vogt for their expert statistical advice.
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Footnotes
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This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 355, B 10), by the Bundesministerium für Bildung, Wissenschaft und Forschung (EC-No. 01EC9407/8) and by a grant from the Faculty of Clinical Medicine Mannheim, University of Heidelberg, Germany.
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